This proposal will examine how the sphingosine 1-phosphate (S1P) regulates smooth muscle cell migration. Our preliminary data show that SMCs of FVB mice migrate well in response to S1P and that migration is inhibited in the presence of a sphingosine kinase inhibitor (SphK). In total contrast, cells of C57BL/6 mice are not stimulated by S1P and the SphK inhibitor actually stimulates migration. We believe these differences are due to S1P receptor expression. FVB express S1P1 and S1P3 but little S1P2. In contrast C57BL/6 cells strongly express S1P2with reduced levels of S1P1 and S1P3. The first aim will measure circulating S1P levels in arteries of FVB and C57BL76 mice before and at times after injury. The activities of the S1P-kinase and S1P-phosphatase and expression of kinases, phosphatase and lyase will also be determined by PCR. S1P will also be determined in lipoprotein and in lipoprotein depleted fractions of plasma. We will also determine if plasma S1P can stimulate events in arteries and importantly affect SMC migration. The second aim will determine if loss of SphK activity influences SMC migration in injured mice arteries. These studies will use SphK null mice as well as a new inhibitor to block SphK activity. The third aim will determine the importance of specific S1P receptors for SMC to migrate. Experiments will be carried out using siRNA to S1P1, S1P2 and S1P3, and S1P2 and S1P3 null cells. The arteries of these mice will be subjected to injury and SMC migration and neointimal development measured. The final aim will determine how S1P signals and so regulates migration. These studies will ask if S1P signals via EGF receptor and if different S1P act differently. We will also determine if PKB and Rac are necessary for SMC migration and if blockade to the EGF receptor will prevent SMC migration and neointimal development in injured arteries.